A polypeptide capable of inhibiting the aggregation of Alzheimer's disease Aβ protein and its application
A technology for use and symptomatic drugs, applied in the field of medicine, can solve the problems of unclear drugs for Alzheimer's disease, insignificant effects, large drug toxicity and side effects, etc., and achieve the effect of broad medical application prospects.
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Embodiment 1
[0044] Example 1. Thiothioflavin T experiment of polypeptide VQVG and Aβ40
[0045] The protein Aβ40 was synthesized by Shanghai Qiangyao Company with a purity greater than 95%. The polypeptide VQVG was synthesized by Shanghai Qiangyao Company, with a purity greater than 95%, and its structural form is unmodified VQVG. Take 1mg each of Aβ40 and polypeptide VQVG, and dissolve them completely with 100ul dimethyl sulfoxide respectively. Thioflavin T (Thioflavin T) was purchased from Sigma Company (product number T3516-5g), with a molecular weight of 318.86. Weigh 0.0319g ThT and dissolve it in 20ml 20mM Tris-HCl (pH 7.5) solution to obtain 5mM ThT mother solution.
[0046] As shown in Table 1, 100ul of the experimental group solution was prepared with pH 7.4 and 20mM Tris-HCl, so that the final concentration of Thioflavin T (Thioflavin T) was 25uM, the final concentration of protein Aβ40 was 5uM, and the final concentration of polypeptide VQVG was 0.5uM. The prepared solution ...
Embodiment 2
[0050] Example 2. Thiothioflavin T experiment of polypeptide VQVG and Aβ42
[0051] Peptide Aβ42 is also synthesized by Shanghai Qiangyao Company, with a purity greater than 95%. The preparation method refers to peptide Aβ40. The sources and formulations of polypeptide VQVG and thiothiaflavin T were consistent with those described in Experimental Example 1. As shown in Table 2, 100 ul of each experimental group solution was prepared with reference to Experimental Example 1. The prepared solution was added to a 96-well plate, placed in a microplate reader, incubated at 37°C, shaken every 5 minutes, and the fluorescence intensity was detected after 48 hours. Each sample was repeated three times and the average value was taken. During the detection, the wavelength of 430nm is used for excitation, and the fluorescence intensity at the wavelength of 485nm is detected, so as to determine the influence of the polypeptide VQVG on the formation of the β-amyloid fibril-like structure....
Embodiment 3
[0055] Example 3 Study on the Effect of Polypeptide VQVG on the Aggregation of Protein Aβ40 at the Cell Level
[0056] In order to detect the effect of the polypeptide VQVG on the Aβ1-40 protein in vivo, the gene corresponding to Aβ40 was constructed in the pEGFP-N1 vector (purchased from Clontech Company) through the restriction site XhoI / BamHI. HEK293T cells were transfected with the correct sequenced plasmid, stimulated with 10uM and 50uM polypeptide VQVG respectively, and cultured at 37°C. At the same time, a group of blank controls were set up, without any stimulation, to grow naturally. After 24 hours, fix with 4% paraformaldehyde, punch holes with 0.3% Triton X-100, and stain nuclei with DAPI. Use confocal (high-sensitivity laser scanning confocal microscope, model Zeiss LSM710) to observe the experimental results, the observation results are as follows: image 3 . It can be seen from the figure that compared with the control group, the intensity of the fluorescent sp...
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